Project Summary: Influenza A virus is a paradigm for understanding viral entry of host cells via receptor-mediated endocytosis. We seek to deepen our understanding of viral membrane fusion in order to identify possible strategies for interfering with the process and arresting the infection cycle. The hemagglutinin (HA) protein mediates fusion of the virus and host cell membranes. HA shares core elements with other type-1 fusion proteins such as those found in Ebola virus and HIV. While certain facets of HA-mediated fusion have been characterized, significant gaps remain in the characterization of the molecular conformations that actually drive membrane fusion, in our understanding of how HA changes conformation, and in our understanding of the interplay between fusion protein and membrane deformation. We propose to use solution X-ray and neutron scattering with ab initio shape reconstruction to determine the structure and dynamics of the hemagglutinin assembly both for isolated HA and HA that is interacting with lipid bilayers. By docking known high resolution structures of components into the solution scattering-derived shape envelopes, pseudoatomic models will be composed. This type of integrative approach is necessary for characterization of complex macromolecular machines. The experiments will provide unique insight into the conformational trajectory traversed by HA as it converts from metastable to fusion-active form, determine whether membrane-bound HA exhibits a propensity to assemble into pore complexes, and elucidate resultant changes in membrane structure. In addition, single-particle fluorescence microscopy will be used to reveal the functional context of HA when it is arrayed on the viral membrane. This approach will identify whether the hundreds of HA spikes on the virus surface become fusion-active in a cooperative fashion. [unreadable] [unreadable] Relevance: Influenza virus is a major human pathogen that in a typical year infects 5-20% of the population of the United States and results in more than 200,000 hospitalizations and approximately 36,000 deaths. The proposed research seeks to gain a detailed understanding of the function of the hemagglutinin machinery that influenza uses to invade new host cells. Such an understanding is necessary in order to identify how best to interfere with the mechanism and to arrest the cycle of viral infection. [unreadable] [unreadable] [unreadable]